The src family of non-receptor tyrosine kinases are a well-studied class of signaling molecules which heretofore have not been shown to play any role in mesoderm induction. All src-family proteins contain catalytic, tyrosine kinase domains, as well as src-homology 2 and src-homology 3 (SH2 and SH3) domains. These domains were first discovered in cytoplasmic (non-receptor) protein tyrosine kinases such as the src oncogene product, thus leading to the term `src homology domains` Sadowski et al., Mol. Cell. Biol. 6:4396-4408 (1986)!. src is the prototypical member of this gene family, and was the first characterized proto-oncogene: elimination of a negative regulatory tyrosine at the carboxy-terminus of src converts the molecule to a potent transforming agent, and all members of the family share this residue Brown and Cooper, Biochimica et Biophysica Acta, 1287:121-149 (1996)!. Nine src-related genes have been cloned thus far, and several of these have been shown to be required for normal function of the immune and nervous systems Brown and Cooper, Biochimica et Biophysica Acta, 1287:121-149 (1996)!. No definitive role, however,. has been described for these proteins during early development.
SH2 and SH3 domains are two individual protein modules that play an intermediary role in eukaryotic cellular signal transduction. After the initiation of the signal by the binding of an extracellular ligand to a transmembrane receptor having an associated tyrosine kinase, SH2 and SH3 domains mediate many of the protein-protein interactions that are necessary for transmission of the signal Cantley et al., Cell, 64:281-302 (1991); Schlessinger et al., Neuron, 9:383-391 (1992); Pawson et al., Curr. Biol., 3:434-442 (1993)!.
The unique importance of these domains became clear with the discovery of the crk oncogene product, which consists of little more than an SH2 and an SH3 domain fused to the viral gag protein, but is capable of transforming cells Mayer et al., Nature, 332:272-275 (1988)!. SH2 and SH3 domains have been identified in molecules with distinct functions that act downstream from the receptors for, among others, epidermal growth actor (EGF), platelet-derived growth factor (PDGF), insulin and interferon, and the T-cell receptor Koch et al., Science; 252:668-674 (1991)!.
The key aspect of the function of SH2 and SH3 domains is their ability to recognize particular amino acid sequences in their target proteins: SH2 domains bind tightly to phosphorylated tyrosine residues Anderrson et al., Science; 250:979-982 (1990); Matsuda et al., Science 248:1537-1539 (1990); Moran et al., Proc. Natl. Acad. Sci. USA 87:8622-8626 (1990); Mayer et al., Proc. Natl. Acad. Sci. USA; 88:627-631 (1991); Songyang et al., Cell 72:767-778 (1993)! whereas SH3 domains bind to unmodified peptide sequence that are rich in proline and hydrophobic amino acids Cicchetti et al., Science 257:803-806 (1992); Ren et al., Science 259:1157-1161 (1993)!. The modular nature of these domains is made clear by the fact that they occur in different positions in the polypeptide chains of the intact proteins of which they are a part, and that the binding functions can often be reproduced by isolated domains. As indicated above, however, the role of src-family tyrosine kinases in the signalling pathway during embryonic development has been obscure.
During embryogenesis, inductive interactions among cells underlie the development of much of the body plan. The process of mesoderm formation is a critical and well-characterized example of an early inductive event. In Xenopus laevis, factors secreted from the vegetal pole induce mesoderm in the adjacent marginal zone Klein and Melton, Endocr. Rev., 15:326-341 (1994)!. During mesoderm induction, it is essential that information received by the marginal zone cells be communicated from the cell surface to the nucleus, where determination of cell fate is driven by an alteration in gene expression. Members of both the Transforming Growth Factor-b (TGF-b) and Fibroblast Growth Factor (FGF) ligand families appear to play essential roles in the formation of mesoderm Klein and Melton, Endocr. Rev., 15:326-341 (1994)!. The downstream effectors of these growth factors are distinct: TGF-b ligands signal through serine-threonine kinase receptors, whose effects are mediated through the recently characterized Smad proteins Massague et al., TICB, 7:187-192 (1997)!. The Smad pathway appears to be quite direct: although a number of positively and negatively acting Smads may interact in mesoderm induction, few other factors seem to be involved in the propagation of signal from cell surface to nucleus. Signaling through the FGF receptor tyrosine kinase, however, appears to be significantly more complex, involving a multiprotein interaction at the plasma membrane, and subsequent activation of the ras/MAP kinase pathway Labonne and Whitman, Dev. Biol., 183:9-20 (1997), and references therein).
The importance of isolating and identifying the factors involved in early development cannot be emphasized. For example, screening for mutations of such factors, in utero, can serve as a powerful tool in early identification of developmental defects. Therefore, there is a need to isolate and identify factors that mediate the signaling initiated at the FGF receptor. Furthermore, there is need to obtain nucleic acid probes and antibodies which can be used to identify the absence of such factors and/or defects in such factors.
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